SN/T 5228.8-2019Rapid screening method for pathogenic microorganisms in exported foods MALDI-TOF MS method Part 8: Klebsiella pneumoniae (English PDF)

Foreword

SN/T 5228-2019 "The MALDI-TOF MS Method for Rapid Screening of Pathogenic Microorganisms in Exported Foods" is divided into 9 parts.

--Part 1: Vibrio alginolyticus;

--Part 2: Clostridium perfringens;

--Part 3: Staphylococcus aureus;

--Part 4: Cronobacterium;

--Part 5: Vibrio vulnificus;

--Part 6: Bacillus cereus;

--Part 7: Campylobacter jejuni;

--Part 8: Klebsiella pneumoniae;

--Part 9: Pseudomonas aeruginosa.

This part is part 8 of SN/T 5228-2019.

This part was drafted in accordance with the rules given in GB/T 1.1-2009.

This part is proposed and managed by the General Administration of Customs of the People's Republic of China.

Drafting organizations of this section. Hangzhou Customs of the People's Republic of China, Beijing Customs of the People's Republic of China.

1 Scope

This part of SN/T 5228 specifies the MALDI-TOF MS rapid screening method for Klebsiella pneumoniae in exported food.

This section applies to the rapid screening of Klebsiella pneumoniae in exported food.

2 Normative references

The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this article Pieces. For undated reference documents, the latest version (including all amendments) is applicable to this document.

GB/T 6682 Analytical laboratory water specifications and test methods GB/T 14926.13 Test method for Klebsiella pneumoniae in laboratory animals GB 19489 Laboratory Biosafety General Requirements

3 Abbreviations

The following abbreviations apply to this document.

BTS. bacterial test standard.

CHCA. alpha-cyano-4-hydroxy-cinnamic acid (alpha-cyano-4-hydroxy-cinnamic acid).

MALDI-TOF MS. matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry).

4 Biosecurity measures

In order to protect the safety of laboratory personnel, Klebsiella pneumoniae should be tested by qualified personnel, and all cultures and waste should be tested for Klebsiella pneumoniae.

Discarded materials should refer to the relevant regulations in GB 19489.

5 Method summary

MALDI-TOF MS is a technology applied to the rapid detection of microbial whole cells, mainly based on the fingerprint analysis of microbial characteristic protein.

Complete the identification and classification of microorganisms. Place the sample of the colony to be identified and an appropriate amount of matrix solution on the sample plate, and form after the solvent volatilizes the co-crystallization of the sample and the matrix, the laser is used as the energy source to radiate the co-crystal, the matrix molecules absorb energy and desorb the sample and make the sample Product ionization, after the time-of-flight analyzer, separates the ions of different mass-to-charge ratios to form a microbial-specific mass spectrum. Will be tested Comparing the substance spectrum with the standard protein fingerprint database of known microorganisms, the species of microorganisms can be determined, and the species of microorganisms can be determined.

是identification.

6 Reagents and materials

Except for special instructions, all reagents are chromatographically pure reagents. The experimental water meets the requirements of first-grade water in GB/T 6682.

6.1 Blood agar plate.

6.2 Buffered peptone water (BPW). see Appendix A, Chapter A.1.

6.3 Cholestyrol Agar (DHL). See Appendix A, Chapter A.2.

6.4 Acetonitrile (ACN).

6.5 Anhydrous ethanol.

6.6 Formic acid.

6.7 Trifluoroacetic acid (TFA).

6.8 CHCA.

6.9 BTS standard product.

6.10 Solvent I. Prepared according to the ratio of water. acetonitrile (6.4). trifluoroacetic acid (6.7) to 50.47.5.2.5 (volume ratio).

Now use.

6.11 Preparation of BTS standard solution. Dissolve BTS standard (6.9) with 50 µL solvent I (6.10). Pipette repeatedly to dissolve BTS powder (avoid vigorous shaking), leave it at room temperature for 5 minutes, and dissolve again. After all BTS (6.9) is dissolved, centrifuge it immediately and use 200 µL PCR tubes are aliquoted, 5 µL per tube, stored at -20°C for later use.

6.12 Preparation of CHCA matrix solution. Add solvent I (6.10) to CHCA (6.8) powder to make the final concentration of CHCA 10 mg/mL.

Shake and mix until the solution is clear. Use 1.5 mL centrifuge tubes for aliquots, 50 µL per tube, seal the mouth of the tube with a parafilm, and store at room temperature for later use.

After storage, if a large amount of precipitation occurs, it should be discarded.

7 Main instruments and equipment

7.1 Bench-top centrifuge. the maximum centrifugal force is >= 16 000 g.

7.2 Vortex oscillator.

7.3 Micro-adjustable pipettes and sterile tips. 2 µL, 100 µL,.200 µL, 1000 µL.

7.4 Matrix-assisted laser desorption ionization time-of-flight mass spectrometer.

8 Detection steps

9 Results judgment and report

9.1 The suspicious colony is identified as Klebsiella pneumoniae by MALDI-TOF MS, and the matching score is greater than or equal to 1.700, the test result can be determined If the result is suspicious, it should be confirmed and reported according to the method of GB/T 14926.13.

9.2 For results other than 9.1, the test result can be determined to be negative, and no Klebsiella pneumoniae is reported.

10 Waste treatment and pollution prevention measures

The waste in the testing process needs to be autoclaved at 121°C for at least 30 minutes before being disposed of.

Appendix A

(Normative appendix) Media and reagents A.1 Buffered peptone water (BPW) A.1.2 Preparation method Heat and dissolve the ingredients in A.1.1, adjust the pH to 7.2±0.2 at 25°C, and dispense into 500 mL jars, each with 225 mL, Autoclave at 121°C for 15 minutes.

A.2 Cholestyrol Agar (DHL) A.2.2 Preparation method in addition to neutral red and agar, add other ingredients to 400 mL of distilled water, stir evenly, let stand for about 10 minutes, heat and boil until completely dissolved. Adjust the pH to 7.3±0.1, then add the agar to 600 mL of distilled water, stir evenly, let stand for about 10 minutes, add Bring to a boil until completely dissolved. After mixing the two solutions, add 6 mL of 5 g/L neutral red aqueous solution, stir evenly, and wait to cool to 50°C ~55°C, pour into a plate for use